JP4499362B2 - Use of neublastin polypeptide to treat neuropathic pain - Google Patents

Description

(Field of Invention)
The present invention relates to the treatment of neuropathic pain (including tactile allodynia) and treatments to reduce the loss of pain sensitivity associated with neuropathy.

(Background of the Invention)
Neuropathic pain is a category of pain that includes several forms of chronic pain and forms that result from nerve dysfunction rather than body tissue. Neuropathic pain (pain resulting from central or peripheral dysfunction) can also be the result of damage to a region of the peripheral or central nervous system, can result from disease, or can be idiopathic . Symptoms of neuropathic pain include burning, tingling, electric sensation, pin and needle sensation, stiffness, limb numbness, kinking sensation, allodynia (usually induced by harmless skin irritation Pain), hyperalgesia (abnormal sensitivity to pain), and hyperpathia (excessive pain response that lasts long after the pain stimulus ceases).

  Some common causes of neuropathic pain are diabetes, cancer chemotherapy, herpes zoster infection, cervical or lumbar compression due to degenerative spinal disease, malignant lesions of the plexus or nerve root, neurodegeneration (eg. , By amputation), HIV infection, and lesions of the central pain pathway (including spinal thalamic tract, thalamus, or thalamus ray). Additional causes of neuropathic pain include drug-induced neuropathy or toxin-induced neuropathy. For example, antiviral agents such as ddI, ddC and d4T generally cause peripheral neuropathy and include phenytoin (anticonvulsant), isoniazid (tuberculosis agent), vincristine, vinblastine, taxol, taxotere and cisplatin The same applies to (cancer chemotherapeutic agents), high-dose vitamins, and folic acid antagonists.

  Current therapies for the management of neuropathic pain are of limited benefit to many patients and include unwanted side effects or dose limiting toxicities. Furthermore, current treatments are symptomatic and do not ameliorate the disease. There remains a need for improved therapies for neuropathic pain management and treatment, particularly those that target the underlying pathology.

(Summary of the Invention)
The present invention provides improved methods for treating neuropathic pain, improved methods for treating tactile allodynia, and improved methods for reducing loss of pain sensitivity associated with neuropathy To do. The methods of the invention employ a neublastin ("NBN") polypeptide, which includes a full-length neublastin polypeptide or a biologically active truncated neublastin polypeptide, These include, for example, at least SEQ ID NOs: 2, 4, 5, and 11-27. Furthermore, the present invention provides a pharmaceutical composition comprising a full length neublastin polypeptide or a truncated neublastin polypeptide suspended, dissolved or dispersed in a pharmaceutically acceptable carrier. .

In certain embodiments, the neublastin polypeptide, SEQ ID NO: 2 _AA -80 _{~AA 140,} SEQ ID NO: 2 _AA -41 _{~AA 140,} SEQ ID NO: 2 of _AA 1 _{~AA 140, AA 25} of SEQ ID NO: 2 ～AA _140, SEQ ID NO: 2 _{AA 28 ~AA 140, AA -80 ~AA} 144 of SEQ ID NO: _{4, AA} 1 ~AA ₁₄₄ of SEQ ID NO: _{4, AA} 1 ~AA ₂₂₄ of SEQ ID NO: 5 or SEQ ID NO: 5, AA ₈₁ ~AA one of ₂₂₄ polypeptide; include C-terminal sequence of any one of SEQ ID NO: 2 of the _AA 107 _{~AA 140} or SEQ ID NO: 2 of the _AA 76 _{~AA 140,} and GDNF family and TGF-beta At least one polypeptide retaining the characteristics of the seven Cys residues of the superfamily; SEQ ID NO: 14 SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: At least one polypeptide comprising number 27; or at least one polypeptide sequence having greater than 70% amino acid homology to the sequences listed above.

  The neublastin polypeptide can be modified with a derivative moiety to have an extended residence time or increased concentration in the body. The neublastin polypeptide can be an N-glycosylated neublastin polypeptide. Furthermore, the neublastin polypeptide can be derivatized with one or more moieties, including a polyethylene glycol moiety, an aliphatic ester, an amide, an N-acyl derivative, or an O-acyl derivative. It is not limited to.

  In one embodiment, the invention features a method for treating neuropathic pain in a subject comprising administering to the subject an effective amount of a neublastin polypeptide (eg, SEQ ID NOs: 2, 4, 5), or an effective amount of an analgesia-inducing compound selected from the group consisting of the following: is also administered to the subject. Occasionally: opioids, antiarrhythmic drugs, local analgesics, local anesthetics, antispasmodics, antidepressants, corticosteroids, and nonsteroidal anti-inflammatory drugs (NSAIDS). In a preferred embodiment, the analgesia-inducing compound is an antispasmodic agent. In another preferred embodiment, the analgesia-inducing compound is gabapentin ((1-aminomethyl) cyclohexaneacetic acid) or pregabalin (S-(+)-4-amino-3- (2-methyl). Propyl) butanoic acid).

  In another embodiment, the invention features a method for treating tactile allodynia in a subject, comprising: an effective amount of a neublastin polypeptide (eg, SEQ ID NOs: 2, 4, 5, and 11-11). An effective amount of a neublastin polypeptide, either alone or with an effective amount of an analgesia-inducing compound selected from the group consisting of: By administration: opioids, antiarrhythmic drugs, local analgesics, local anesthetics, antispasmodics, antidepressants, corticosteroids, and NSAIDS. In a preferred embodiment, the analgesia-inducing compound is an antispasmodic agent. In another preferred embodiment, the analgesia-inducing compound is gabapentin ((1-aminomethyl) cyclohexaneacetic acid) or pregabalin (S-(+)-4-amino-3- (2-methylpropyl) butanoic acid). It is.

  The neublastin polypeptide can be administered concomitantly with a therapeutic agent, which includes but is not limited to anti-cancer or anti-viral agents. Anti-cancer agents include but are not limited to taxol, taxotere, cisplatin, nocodazole, vincristine, vindesine and vinblastine. Antiviral agents include, but are not limited to, ddI, DDC, d4T, foscanet, dapsone, metronidazole, and isoniazid.

  The present invention encompasses a method for treating neuropathic pain in a subject. In certain embodiments, the neuropathic pain is associated with diabetic neuropathy. In another embodiment, the neuropathic pain is associated with infection of a subject by a virus, which includes, but is not limited to, herpes virus, human immunodeficiency virus (HIV), and papilloma virus. . Neuropathic pain can be associated with herpes zoster virus infection, or in particular postherpetic neuralgia. In a further embodiment, the neuropathic pain is associated with sciatica. In another embodiment, the invention features a method for modulating loss of pain sensitivity in a subject suffering from neuropathy. In a preferred embodiment, the neuropathy is diabetic neuropathy. In another preferred embodiment, the loss of pain sensitivity is a loss of thermal pain sensitivity.

  In further embodiments, the neuropathic pain is hyperalgesia, phantom pain, thermal hyperalgesia, or due to injury associated with trauma. In addition, neuropathic pain may also be associated with: hereditary neuropathy (including but not limited to Fleetrich ataxia, familial amyloid polyneuropathy, Tangier disease, Fabry disease), metabolic disorders (renal Insufficiency and hypothyroidism), vitamin deficiency (including but not limited to vitamin B12 deficiency, vitamin B6 deficiency, and vitamin E deficiency), toxic neuropathy and iatrogenic neuropathy ( Alcoholism, Vitamin B6 poisoning, Hexacarbon poisoning, Amiodarone, Chloramphenicol, Disulfiram, Isoniazid, Gold, Lithium, Metronidazole, Misonidazole, Nitro Including, but not limited to, run-in), infectious neuropathy (including but not limited to leprosy, Lyme disease), autoimmune neuropathy (Gian-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, important Unspecified monoclonal hypergammaglobulinemia, and multiple neuropathies), trigeminal neuralgia, entrapment syndrome (including but not limited to carpal tunnel), trauma Posterior neuralgia, phantom limb pain, multiple sclerosis pain, complex local pain syndrome (including but not limited to reflex sympathetic dystrophy, causalgia), neoplasia, vasculitis / vasculopathy neuropathy And idiopathic neuropathy.

  The foregoing method contemplates administering a formulation containing a neublastin polypeptide to a subject, preferably systemically, at a dosage between 1 μg and 30,000 μg per kg body weight of the subject per dose. To do. In an alternative embodiment, the dosage is between 10 μg and 30,000 μg per kg body weight of the subject per dose; between 10 μg and 10,000 μg per kg body weight of the subject per dose; Between 25 μg and 10,000 μg per kg body weight of the subject; between 25 μg and 3000 μg per kg body weight of the subject per dose; and between 50 μg and 3000 μg per kg body weight of the subject per dose Between.

  The neublastin polypeptide used in the foregoing methods can be administered via any suitable delivery system and preferably from intravenous delivery, intramuscular delivery, intrapulmonary delivery, subcutaneous delivery, and intraperitoneal delivery. It can be administered via a delivery system from the group consisting of most preferably via intramuscular or subcutaneous delivery. The neublastin polypeptide used in the foregoing methods can also be administered via intrathecal delivery.

  The NBN polypeptide-containing formulation of the invention can be administered in a timed-released composition.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will follow. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

  Other features and advantages of the invention will be apparent from the following detailed description and from the claims.

(Detailed description of the invention)
The present invention relates to methods and compositions for treating neuropathic pain, tactile dysfunction, by administering a neuroblastin polypeptide to a subject at risk of neuropathic pain or a subject suffering from neuropathic pain. It relates to methods and compositions for treating tactile allodynia, and methods and compositions for reducing loss of pain sensitivity.

  Neublastin polypeptides are proteins that promote survival, maintain phenotypic differentiation, prevent degeneration, promote regeneration, and restore the activity of neuronal cells and tissues. Alternatively, neublastin (e.g., first described in WO 00/01815) is referred to as "artemin" (see, e.g., WO 00/18799) and "enovin" (e.g., WO 00 / 040050). Neublastin has been classified as a distant member of the TGF-β superfamily (Massague et al., 1994, Trends in Cell Biology, 4: 172-178), and GDNF, parsephin (“PSP”; Milbrandt et al. 1998, Neuron 20: 245-253 (incorporated herein by reference)) and neurturin ("NTN"; WO 97/08196 (incorporated herein by reference)). A member of the glial cell line-derived neurotrophic factor ligand family (“GDNF”; WO 93/06116 (incorporated herein by reference)) within the family that it contains. GDNF subfamily ligands have in common the ability to induce signaling through the RET receptor tyrosine kinase. These three ligands of the GDNF subfamily differ in relative affinity for the GFRα receptor, a family of neurotrophic receptors. Neublastin preferably acts through the GFRα3-RET complex. Baudet et al., Development, 127, 4335-44 (2000); Baloh et al., Neuron, 21, 1291-1302 (1998); Airaksinen et al., Mol. Cell. Neuroscience, 13, pp. 313-325 (1999).

  Table 1 shows the amino acid sequence comparison of neublastin (SEQ ID NO: 2) against GDNF subfamily members Neuturin (SEQ ID NO: 6), Percefin (SEQ ID NO: 7) and GDNF (SEQ ID NO: 8). Neublastin polypeptides useful in the present invention preferably retain the GDNF subfamily fingerprint (ie, the underlined amino acid residues in Table 1).

表１に示されるアミノ酸配列整列から、ニューブラスチンが、ＴＧＦ−βスーパーファミリー内で保存される位置に７つのシステイン残基を有することが見出され得る。この配列整列に基づいて、ニューブラスチンは、神経栄養性因子（ＬＧＬＧ−ＦＲ（Ｙ／Ｆ）ＣＳＧＳＣ−ＱｘＣＣＲＰ−ＳＡｘｘＣＧＣ、ＧＤＮＦサブファミリーフィンガープリント、表１において下線を付す）のＧＤＮＦサブファミリーのメンバーであることが示された。

From the amino acid sequence alignment shown in Table 1, it can be found that neublastin has seven cysteine residues at positions that are conserved within the TGF-β superfamily. Based on this sequence alignment, neublastin is a member of the GDNF subfamily of neurotrophic factors (LGLG-FR (Y / F) CSGSC-QxCCRP-SAxxCGC, GDNF subfamily fingerprint, underlined in Table 1) It was shown that.

Neublastin polypeptides useful herein are pre-pro-protein, pro-protein, mature protein, glycosylated protein, phosphorylated protein, truncated form, or any other post-translationally modified protein It can be provided in any physiologically active form, including Bioactive neublastin is assumed to be a dimerized form for each NBN variant. This is because dimer formation is required for activity. Little or no activity is observed in the monomeric NBN polypeptide. Bioactive neublastin polypeptide binds to GFRα3 or a complex of GFRα3 and RET in the presence of a cofactor (eg, GFRα3 or RET) and induces RET dimerization and RET autophosphorylation A dimerized polypeptide. Thus, as used herein, a “neublastin polypeptide” is a polypeptide possessing neurotrophic activity (eg, as described in WO 00/01815) as follows:
(1. Wild-type neublastin)
The following “wild type” neublastin amino acid (“aa” or “AA”) sequences are examples of those useful in the methods and compositions of the invention:
- SEQ ID NO: 2 of the _AA -80 _{-AA 140} ( "wild-type" human prepro),
- SEQ ID NO: 2 _AA -41 _{-AA 140} (Purohito)
-AA ₁ -AA ₁₄₀ of SEQ ID NO: 2 (mature 140AA (SEQ ID NO: 11); hereinafter “140NBN”),
-AA ₂₅ -AA ₁₄₀ of SEQ ID NO: 2 (mature 116AA (SEQ ID NO: 12); hereinafter "116NBN"),
- SEQ ID NO: 2 _AA 28 _{-AA 140} (mature 112 aa (SEQ ID NO: 13); "113NBN" hereinafter),
- _AA -80 _{-AA 144} of SEQ ID NO: 4 (murine prepro),
-AA ₁ -AA ₁₄₄ of SEQ ID NO: 4 (mouse matured-144AA),
- _AA 1 _{-AA 224} of SEQ ID NO: 5 (rat prepro),
- _AA 81 _{-AA 224} of SEQ ID NO: 5 (rat mature --144AA),
-7 Cys having a C-terminal sequence as shown in AA ₁₀₇ -AA ₁₄₀ of SEQ ID NO: 2 and more preferably AA ₇₆ -AA ₁₄₀ of SEQ ID NO: 2 and unique to the GDNF family and TGF-β superfamily A peptide that retains residues.

  In one embodiment, a preferred neublastin polypeptide comprises (seven) cysteines conserved at positions 43, 70, 74, 107, 108, 136, and 138 of SEQ ID NO: 2. These seven conserved cysteine residues are composed of three intra-monomer disulfide bonds (eg, between cysteine residues 43-108, 70-136, and 74-138 in SEQ ID NO: 2) and 1 Two intermonomer disulfide bonds (eg, intended in SEQ ID NO: 2 between cysteine residues 107-107) (these are conserved structural for the TGF-β superfamily, along with an extended β chain region) Is known within the TGF-β superfamily. See, for example, Daopin et al., Proteins 1993, 17: 176-192.

(2. Short neublastin ("NBN"))
Neublastin polypeptides useful in the present invention also include truncated forms of full-length neublastin molecules. In such a truncated molecule, one or more amino acids have been deleted from the N-terminus or C-terminus, preferably from the N-terminus. The truncated neublastin polypeptide provides a mature neublastin polypeptide, and the mature neublastin polypeptide is contacted with at least one protease under conditions sufficient to produce a truncated neublastin polypeptide. Can be obtained. Preferably, the at least one protease is an exoprotease and contacting the mature neublastin polypeptide results in the formation of an exopeptidase neublastin polypeptide digestion product that can be further digested with dipeptidyl peptidase.

  The truncated neublastin polypeptides described herein preferably comprise a polypeptide sequence comprising seven cysteine residues that are conserved in the mature neublastin sequence. In certain preferred embodiments, the truncated neublastin polypeptide comprises at least 85 carboxy terminal amino acids of the mature 113NBN neublastin polypeptide.

Other variants of neublastin include a truncated NBN form. Examples of these include the following (i) to (xiv):
(I) a 112AA polypeptide sequence designated herein as NBN112, which is the carboxy-terminal 112 amino acids of the mutated neublastin polypeptide (eg, amino acids 29-140 of SEQ ID NO: 2 (SEQ ID NO: 14)) Have
(Ii) a 111AA polypeptide sequence designated herein as NBN111, which is the carboxy-terminal 111 amino acids of the mutated neublastin polypeptide (eg, amino acids 30-140 of SEQ ID NO: 2 (SEQ ID NO: 15)) Have
(Iii) 110AA polypeptide sequence designated herein as NBN110, which is the carboxy terminal 110 amino acids of the mutated neublastin polypeptide (eg, amino acids 31-140 of SEQ ID NO: 2 (SEQ ID NO: 16)) Have
(Iv) a 109AA polypeptide sequence designated herein as NBN109, which is the 109 amino acids at the carboxy terminus of the mutated neublastin polypeptide (eg, amino acids 32-140 of SEQ ID NO: 2) Have
(V) a 108AA polypeptide sequence designated herein as NBN108, which is the carboxy-terminal 108 amino acids of the mutated neublastin polypeptide (eg, amino acids 33-140 of SEQ ID NO: 2 (SEQ ID NO: 18)) Have
(Vi) a 107AA polypeptide sequence designated herein as NBN107, which is the 107 amino acids at the carboxy terminus of the mutated neublastin polypeptide (eg, amino acids 34-140 of SEQ ID NO: 2 (SEQ ID NO: 19)) Have
(Vii) a 106AA polypeptide sequence designated herein as NBN106, which is the 106 amino acids at the carboxy terminus of the mutated neublastin polypeptide (eg, amino acids 35-140 of SEQ ID NO: 2 (SEQ ID NO: 20)) Have
(Viii) 105AA polypeptide sequence designated herein as NBN105, which is the carboxy-terminal 105 amino acids of the mutated neublastin polypeptide (eg, amino acids 36-140 of SEQ ID NO: 2 (SEQ ID NO: 21)) Have
(Ix) 104AA polypeptide sequence designated herein as NBN104, which is the 104 amino acids at the carboxy terminus of the mutated neublastin polypeptide (eg, amino acids 37-140 of SEQ ID NO: 2 (SEQ ID NO: 22)) Have
(X) a 103AA polypeptide sequence designated herein as NBN103, which is the 103 amino acids at the carboxy terminus of the mutated neublastin polypeptide (eg, amino acids 38-140 of SEQ ID NO: 2 (SEQ ID NO: 23)) Have
(Xi) a 102AA polypeptide sequence designated herein as NBN102, which is the carboxy-terminal 102 amino acids of the mutated neublastin polypeptide (eg, amino acids 39-140 of SEQ ID NO: 2 (SEQ ID NO: 24)) Have
(Xii) a 101AA polypeptide sequence designated herein as NBN101, which is the 101 amino acids at the carboxy terminus of the mutated neublastin polypeptide (eg, amino acids 40-140 of SEQ ID NO: 2 (SEQ ID NO: 25)) Have
(Xiii) a 100AA polypeptide sequence designated herein as NBN100, which is the carboxy terminal 100 amino acids of the mutated neublastin polypeptide (eg, amino acids 41-140 of SEQ ID NO: 2 (SEQ ID NO: 26)) Have
(Xiv) a 99AA polypeptide sequence designated herein as NBN99, which is the 99 amino acids at the carboxy terminus of the mutated neublastin polypeptide (eg, amino acids 42-140 of SEQ ID NO: 2 (SEQ ID NO: 27)) Have

  It is understood that the truncated forms of neublastin disclosed herein (e.g., 112AA-99AA forms) have neurotrophic activity.

  In the most preferred embodiment, the truncated neublastin polypeptide is amino acids 99aa, 100aa, 101aa, 102aa, 103aa, 104aa, 105aa, 106aa, 107aa, 108aa, 109aa, 110aa at the carboxy terminus of the mutant 113AA neublastin polypeptide. , 111aa, or 112aa (ie, NBN99, NBN100, NBN101, NBN102, NBN103, NBN104, NBN105, NBN106, NBN107, NBN108, NBN109, NBN110, NBN111, or NBN112, respectively). These sequences are also represented as mouse and rat as 99aa, 100aa, 101aa, 102aa, 103aa, 104aa, 105aa, 106aa, 107aa, 108aa, 109aa, 110aa, 111aa, or 112aa, respectively, in SEQ ID NOs: 4 and 5. Can also be found in other neublastin polypeptides. These most preferred examples of truncated NBN forms are bioactive (referred to herein as “bioactive shortened neublastin peptides”) when shown herein to have neurotrophic activity. As noted above, NBN dimers are required for bioactivity when little or no activity is observed with NBN monomeric polypeptides.

(3. A substantially similar or identical mutant neublastin ("NBN"))
NBN useful in the present invention also includes those NBN polypeptides having amino acid sequences substantially similar or identical to the various prepro, pro, mature, and truncated “neublastin” polypeptides described above. Preferably, the neublastin polypeptide used is at least 70%, more preferably 85%, even more preferably 90%, relative to the neublastin polypeptide of SEQ ID NO: 2, 4, 5 or 11-27, or Even more preferably, it has 95% identity or similarity. Most preferably, the neublastin polypeptide used has at least 99% identity or similarity to the neublastin polypeptide of SEQ ID NO: 2, 4, 5 or 11-27.

  The degree to which a candidate polypeptide shares homology with a neublastin polypeptide of the invention is determined as the degree of similarity or identity between the two amino acid sequences.

  A high level of sequence identity indicates that the first sequence may be derived from the second sequence. Amino acid sequence identity requires an identical amino acid sequence between two aligned sequences. Thus, a candidate sequence that shares 70% amino acid identity with a reference sequence requires that, after alignment, 70% of the amino acids in the candidate sequence are identical to the corresponding amino acid sequence in the reference sequence. Identity is determined by computer analysis, for example, but not limited to, the ClustalX computer alignment program (Thompson JD, Gibson TJ, Plewanak F, Jeanouginfin F, and Higgins lenflece lenflece lenfle sig lensf lend sig lens lenf stigf s by quality analysis tools ", Nucleic Acids Res. 1997, 25 (24): 4876-82), and the default parameters suggested herein. Using this program, the mature portion of the polypeptide encoded by the analog DNA sequence of the invention can be expressed as SEQ ID NO: 2 (human NBN), SEQ ID NOs: 4 and 5 (rodents), or SEQ ID NOs: 11-27 ( At least 70%, more preferably 85%, even more preferably 85%, even more preferably 90%, or even more preferably with the amino acid sequences provided herein, such as mature NBN and truncated NBN) Indicates a degree of identity of 95%, most preferably at least 99%.

  Other alignment tools are known and are described, for example, in Needleman et al. Mol. Biol. 48: 443 (1970), and Align Program (a commercial software package manufactured by DNAstar, Inc.), the teachings of which are incorporated herein by reference. . Once the alignment between the candidate sequence and the reference sequence is made and refined, a percent homology score is calculated. The individual amino acid sequences of each sequence are compared sequentially according to their similarity to each other.

  Similarity factors include similar size, shape and charge. One particularly preferred method of determining amino acid similarity is described in Dayhoff et al., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE 345-352 (1978 and addendum), which is incorporated herein by reference. PAM 250 matrix. The similarity score is first calculated as the sum of an aligned set of amino acid similarity scores. Insertions and deletions are ignored for purposes of percent homology and identity. Therefore, no gap penalty is used for this calculation. The raw score is then normalized by dividing the raw score by the geometric mean of the neublastin polypeptide candidate compound and the seven cysteine backbone scores. This geometric mean is the square root of the product of these scores. This normalized raw score is percent homology.

  As described above, the neublastin polypeptides of the present invention include variant polypeptides. In the context of the present invention, the term “variant polypeptide” is provided as SEQ ID NO: 2 (human NBN), SEQ ID NOs: 4 and 5 (rodents), or SEQ ID NOs: 11-27 (mature NBN and truncated NBN). A polypeptide (or protein) having an amino acid sequence that differs from one sequence at one or more amino acid positions. Such variant polypeptides include modified polypeptides as described above, as well as conservative substitutions, splice variants, isoforms, homologs from other species, and polymorphisms.

  As defined herein, the term “conservative variant” refers to the replacement of an amino acid residue by another biologically similar residue. Typically, the biological similarity as mentioned above reflects the replacement of the wild type sequence with a conserved amino acid. For example, conservative amino acid substitutions with little or no effect on biological activity are to be expected, particularly if the substitution represents less than 10% of the total amount of residues in the polypeptide or protein. Preferably, conservative amine acid substitutions show a change of less than 5% of the polypeptide or protein, most preferably less than 2% of the polypeptide or protein. For example, when calculated according to human 113NBN, the most preferred conservative substitutions show fewer than three amino acid substitutions in the wild type mature amino acid sequence. In particularly preferred embodiments, there is a single amino acid substitution in the mature sequence. Here, both substituted or exchanged amino acids are acyclic.

  Other examples of specific conservative substitutions include substitution of one hydrophobic residue for another (isoleucine, valine, leucine, or methionine), or other polar residues of one polar residue Substitution to a group (eg, substitution of arginine with lysine, substitution of glutamic acid with aspartic acid, or substitution of glutamine with asparagine, etc.) can be mentioned.

  The term conservative substitution also refers to the replacement amino acid residue in place of the parent amino acid residue that is not substituted, provided that the antibody raised against the substituted polypeptide also immunoreacts with the unsubstituted polypeptide. Including use.

  This modification of the primary amino acid sequence can produce a protein that has substantially equivalent activity compared to the corresponding unmodified polypeptide and can therefore be considered a functional analog of this parent protein. . Such modifications can be intentional, such as by site-directed mutagenesis, or they can occur naturally and include splice variants, isoforms, from other species Examples include homologs and polymorphisms. Such functional analogs are also contemplated according to the present invention.

  Furthermore, modification of the primary amino acid sequence can produce proteins that do not retain the biological activity of the parent protein, including dominant negative forms. Dominant negative proteins usually interact with wild-type proteins by binding to regulatory elements that interact functionally with this polypeptide (eg, upstream or downstream components) or otherwise sequestering the regulatory elements. Can interfere. Such dominant negative forms are also contemplated according to the present invention.

(4. Derivatives or modified NBN)
Polypeptides of the invention also include chimeric polypeptides or cleavable fusion polypeptides (another polypeptide is fused to the N-terminus or C-terminus of this polypeptide or fragment thereof). A chimeric polypeptide can be produced by fusing a nucleic acid sequence (or part thereof) encoding another polypeptide to a nucleic acid sequence (or part thereof) of the invention. Techniques for producing chimeric polypeptides are standard techniques. Such techniques usually require ligating the sequences into the same reading frame and expressing the fused polypeptide under the control of the same promoter and terminator.

  The neublastin polypeptide can be an N-glycosylated polypeptide. In one embodiment, the Asn residue at position 122 of SEQ ID NO: 2 is glycosylated.

  The present invention also includes US Pat. Nos. 5,434,131; 5,565,335; 5,541,087; and 5,726,044, each of which is herein incorporated by reference. Contemplated are neublastin fusion proteins (eg, Ig-fusions) as described in, incorporated herein by reference, or preferably serum albumin fusions.

  Neublastin polypeptides useful in the present invention include time-release compositions and neublasts modified with a derivative moiety (preferably a polyethylene glycol moiety) to have an extended residence time and / or increased concentration in body fluids. A blastine polypeptide is mentioned. Further, the neublastin polypeptide can be derivatized with a moiety selected from the group consisting of aliphatic esters, amides, N-acyl-derivatives, or O-acyl derivatives.

Table 2 below shows a Clustal W comparison between human (SEQ ID NO: 2), mouse (SEQ ID NO: 4), and rat (SEQ ID NO: 5) prepro-neublastin. The mature NBN polypeptides NBN140, NBN116, NBN113, and NBN99 are indicated by “ ^* ”. The N-terminus of NBN112 to NBN100 is indicated by a continuous “:” symbol. Table 3 below shows a distance comparison between wild type human, mouse, and rat neublastin polypeptides (SEQ ID NOs: 2, 4, and 5).

（ニューブラスチンポリペプチドを生成する方法）
本明細書中で使用されるニューブラスチンポリペプチドは、哺乳動物細胞（好ましくは、ヒト細胞）またはマウス起源の細胞から単離され得る。最も好ましい実施形態において、ニューブラスチンポリペプチドは、ヒト心臓組織、ヒト骨格筋、ヒト膵臓、またはヒト脳組織から（特に、尾状核または視床から）単離され得るか、あるいは以下でより詳細に記載されるように、哺乳動物起源のＤＮＡから獲得され得る。

(Method for producing neublastin polypeptide)
As used herein, a neublastin polypeptide can be isolated from mammalian cells (preferably human cells) or cells of mouse origin. In the most preferred embodiments, the neublastin polypeptide can be isolated from human heart tissue, human skeletal muscle, human pancreas, or human brain tissue (particularly from the caudate nucleus or thalamus), or in more detail below. As described, it can be obtained from DNA of mammalian origin.

  Alternatively, a neublastin polypeptide can be obtained by expression of a polynucleotide encoding such a neublastin polypeptide. Such polynucleotides include DNA sequences, cDNA sequences, and RNA sequences and are available in the art. See, for example, WO00 / 01815, WO00 / 04050, and WO00 / 18799, which are hereby incorporated by reference. A particularly useful polynucleotide has the DNA sequence shown in SEQ ID NO: 1 (human NBN cDNA) and the DNA sequence shown in SEQ ID NO: 3 (mouse NBN cDNA). In addition, the genomic sequence of human NBN can be used (see GenBank accession number AC005038).

  More particularly, a neublastin polypeptide useful in the present invention comprises culturing cells containing a nucleic acid sequence encoding a neublastin polypeptide under conditions that permit production of the neublastin polypeptide, and then culturing medium. From which the neublastin polypeptide is recovered. The nucleic acid sequence encoding a neublastin polypeptide is usually a nucleic acid sequence that is endogenous to the cell, or can be a foreign nucleic acid sequence that has been introduced into the “producing” cell. If the cell has been genetically modified to produce a neublastin polypeptide, the cell can be modified by conventional methods or gene activation.

  In accordance with conventional methods, DNA molecules comprising neublastin cDNA or genomic DNA sequences can be included in expression constructs and can be standard methods (liposome mediated transfection, polybrene mediated transfection, or DEAE dextran mediated transfection, electro It is transferred into the cells by poration, calcium phosphate precipitation, microinjection, or velocity driven microprojectile ("microparticle gun")). Alternatively, a system that delivers DNA by viral vectors can be used. Viruses known to be useful for gene transfer include adenovirus, adeno-associated virus, lentivirus, herpes virus, mumps virus, poliovirus, retrovirus, Sindbis virus and vaccinia virus (e.g. Canarypox virus), as well as baculovirus infection of insect cells (especially Sf9 insect cells).

  Alternatively, the cell may be a gene activation (“GA”) approach (eg, US Pat. Nos. 5,733,761 and 5,750,376, each incorporated herein by reference). Can be modified to produce a neublastin polypeptide using the approach described in US Pat.

  Thus, the term “genetically modified” as used herein with respect to a cell, introduces a nucleic acid molecule that encodes a regulatory element that controls the expression of the gene product and / or endogenous coding sequence for the gene product. Later, it is meant to include cells that express a particular gene product. Nucleic acid molecules can be introduced by gene targeting, allowing the introduction of nucleic acid molecules at specific genomic sites.

  In one embodiment, the neublastin polypeptide is produced in a bacterial cell (preferably E. coli). In different embodiments, the neublastin polypeptide is produced in insect-derived cells (particularly Sf9).

  In another embodiment, the neublastin polypeptide can be produced, for example, in mammalian cells (eg, human cells), oocytes, or yeast cells. The cells of the present invention may be human embryonic kidney (“HEK”) cells (eg, HEK293 cells, BHK21 cells, Chinese hamster ovary (“CHO”) cells, Xenopus laevis oocytes (“XLO”) cells, or Pichia pastoris ( Yeast))), but is not limited thereto. In one embodiment, the cells of the invention are fungal cells (eg, filamentous fungal cells). In yet another embodiment, the cell is an insect cell, most preferably an Sf9 cell. Another mammalian cell of the present invention is the PC12 cell line, HiB5 cell line, RN33b cell line, human neural progenitor cells, and other cells derived from human cells, particularly nerve cells.

  Examples of primary or secondary cells include fibroblasts, epithelial cells (including breast and intestinal epithelial cells), endothelial cells, blood-forming elements (including lymphocytes and bone marrow cells), glial cells, hepatocytes , Keratinocytes, muscle cells, nerve cells, or precursors of these cell types. Examples of immortalized human cell lines useful in the methods of the present invention include, but are not limited to: Bowes melanoma cells (ATCC accession number CRL 9607), Daudi cells (ATCC accession number CCL 213), HeLa cells and derivatives of HeLa cells (ATCC registration number CCL 2, CCL 2.1 and CCL 2.2), HL-60 cells (ATCC registration number CCL 240), HT-1080 cells (ATCC registration number CCL121), Jurkat cells (ATCC registration number TIB 152), KB cancer cells (ATCC registration number CCL 17), K-562 leukemia cells (ATCC registration number CCL 243), MCF-7 breast cancer cells (ATCC registration number BTH 22), MOLT-4 cells ( ATCC registration number 1582), Na alwa cells (ATCC registration number CRL 1432), Raji cells (ATCC registration number CCL 86), RPMI 8226 cells (ATCC registration number CCL 155), U-937 cells (ATCC registration number CRL 1593), WI-38VA13 subline 2R4 cells (ATCC accession number CLL75.1), and 2780AD ovarian cancer cells (Van der Brick et al., Cancer Res. 48: 5927-5932, 1988), and heterohybridomas produced by fusion of human cells with cells of another species cell. Secondary human fibroblast cell lines such as WI-38 (ATCC accession number CCL 75) and MRC-5 (ATCC accession number CCL 171)) may also be used.

  When the cell is a eukaryotic cell, the integration of the heterologous polynucleotide of the present invention is notably by infection (using a viral vector), by transfection (using a plasmid vector), calcium phosphate precipitation, microinjection, electroporation. , Lipofection, or other physicochemical methods known in the art.

  NBN polypeptides are isolated from producer cell culture or from culture medium conditioned by producer cells, if applicable, using standard protein purification techniques (including refolding). Suitable techniques are described in the examples below.

(Subject for treatment)
The present invention relates to a mammal suffering from or at risk of treating neuropathic pain (including tactile allodynia) and to reduce the loss of pain sensitivity associated with neuropathy. It can be used in animal subjects. Subjects at risk of developing neuropathy and the loss of pain sensitivity associated with such neuropathies include those with diabetes, subjects undergoing chemotherapy, and certain trauma. Subject, subject taking various toxicants or drugs, subject suffering from certain vitamin deficiencies, subject infected with certain viral pathogens, suffering from various autoimmune and metabolic disorders Subjects, as well as subjects who have suffered specific nerve injury or neurodegeneration. Mammalian subjects include sheep, horses, dogs, cats, pigs, rabbits, guinea pigs, rats, hamsters, gerbils and mice, most preferably humans.

  Typically, in a human subject, the patient is refractory to other traditional pain treatments, or the subject has other such pain to provide good pain control. Responds poorly to treatment.

  In general, the invention features both prophylactic and therapeutic treatment protocols. In prophylactic treatment, a neublastin polypeptide is administered to a subject at risk of neuropathic pain or loss of pain sensitivity; such a subject is expected to be a subject with early neuropathy. The Treatment with neublastin under such circumstances is useful for prophylactic treatment of at-risk patients.

  In therapeutic treatment, a neuroblastin polypeptide is administered to a subject suffering from neuropathic pain or a subject suffering from loss of pain sensitivity as a result of neuropathic morbidity; such a subject is treated with late neuropathy Expected to be a subject with Treatment with neublastin under such circumstances helps to alleviate neuropathic pain and / or assist in appropriate pain sensitivity in the subject. Such late patients may have received a number of treatments beginning with self-medication (eg, non-steroidal anti-inflammatory drugs such as ibuprofen). Such treatments include antidepressants (eg, the tricyclic antidepressant vanlafaxine, and selective serotonin reuptake inhibitors (specific mediators include amitriptyline, desipramine, imipramine and nortriptyline)) Or may be escalated to antispasmodic drugs (eg, gabapensin, carbamazepine, lamotrigine, topiramate and phenytoin). Other medicaments include topical analgesics (eg, capsaicin and lidoderm), antiarrhythmic drugs and opioids. In the case of severe neuropathy, surgery can be performed. Studies show that fewer than 50% of patients respond to local analgesics, antiarrhythmic drugs, opioids or surgery.

(Method and pharmaceutical composition)
The present invention provides methods for treating neuropathic pain, treating tactile allodynia, and reducing the loss of pain sensitivity associated with neuropathy. The method of the present invention uses a neublastin polypeptide (including a full-length neublastin polypeptide or a bioactive truncated neublastin polypeptide). Furthermore, the present invention provides a pharmaceutical composition in which a full-length neublastin polypeptide or a truncated neublastin polypeptide is suspended, dissolved or dispersed in a pharmaceutically acceptable carrier.

(1. Treatment of neuropathic pain)
In one embodiment, the invention features a method for treating neuropathic pain in a subject, the method comprising administering to the subject an effective amount of a neublastin polypeptide. The neublastin polypeptide can be administered alone (monotherapy) or can be administered as part of a combination therapy regimen. A preferred combination therapy is an effective amount of analgesia selected from the group consisting of opioids, antiarrhythmic drugs, local analgesics, local anesthetics, antispasmodics, antidepressants, corticosteroids and nonsteroidal anti-inflammatory drugs (NSAIDS). A step of administering an inducible compound to the subject.

  The neublastin polypeptides and neublastin nucleic acids of the invention (and pharmaceutical compositions comprising these polypeptides and nucleic acids described herein) are used in the treatment of pain associated with peripheral neuropathy. . Peripheral neuropathies that can be treated according to the present invention are related to trauma-induced neuropathies (eg, neuropathies caused by physical injury or disease state), physical damage to the brain, physical damage to the spinal cord, brain damage Neurological disorders associated with seizures and neurodegeneration.

  The invention also includes chemotherapy-induced neuropathies (eg, neuropathies caused by delivery of chemotherapeutic agents (eg, taxol or cisplatin)); toxin-induced neuropathies, drug-induced neuropathies, pathogen-induced (eg, virus-induced ) Provides treatment for neuropathy, vitamin deficiency-induced neuropathy; idiopathic neuropathy; and diabetic neuropathy. See, for example, US Pat. Nos. 5,496,804 and 5,916,555, each of which is incorporated herein by reference. The present invention still further treats single neuropathy, single-multiple neuropathy and multiple neuropathy (including axonal and demyelinating neuropathies) using the neuroblastin nucleotides and neublastin polypeptides of the invention. Can be used to

Neuropathic pain can be associated with a number of peripheral neuropathies, including:
(A) trauma-induced neuropathy,
(B) chemotherapy-induced neuropathy,
(C) Toxin-induced neuropathy (alcoholism, vitamin B6 poisoning, hexacarbon toxication, amiodarone, chloramphenicol, disulfiram, isoniazid, gold, lithium, metronidazole, misonidazole, nitrofurantoin But not limited to)
(D) drug-induced neuropathy, including therapeutic drug-induced neuropathic pain (eg, neuropathy caused by an anticancer agent, particularly an anticancer agent selected from the group consisting of taxol, taxotere, cisplatin, nocodazole, vincristine, vindesine and vinblastine; and Antiviral drugs, in particular neuropathies caused by antiviral drugs selected from the group consisting of ddI, DDC, d4T, foscanet, dapsone, metronidazole and isoniazid)),
(E) vitamin deficiency-induced neuropathy (including but not limited to vitamin B12 deficiency, vitamin B6 deficiency, and vitamin E deficiency),
(F) idiopathic neuropathy,
(G) Diabetic neuropathy,
(H) pathogen-induced nerve injury,
(I) inflammation-induced nerve damage,
(J) neurodegeneration,
(K) hereditary neuropathy (including but not limited to Fleetrich ataxia, familial amyloid neuropathy, Tangier disease, Fabry disease),
(L) metabolic disorders (including but not limited to renal failure and hypothyroidism),
(M) Infectious and viral neuropathies (leprosy, neuropathic pain associated with Lyme disease, viruses (especially herpesviruses (eg, herpes zoster that can lead to postherpetic neuralgia), human immunodeficiency virus (HIV) and papillomaviruses) A neuropathic pain associated with infection by a virus selected from the group consisting of),
(N) Autoimmune neuropathies (including, but not limited to, Giant-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, monoclinic hypergammaglobulinemia of unknown significance and polyneuropathy) ,
(O) Trigeminal neuralgia and entrapment syndrome (including but not limited to carpal tunnel),
(P) Other neuropathic pain syndromes (post-traumatic neuralgia, phantom limb pain, multiple sclerosis pain, complex local pain syndromes including but not limited to reflex sympathetic dystrophy, causalgia) , Including neoplasia-related pain, vasculitis / vasopathic neuropathy, and sciatica).

  Neuropathic pain can manifest as allodynia, hyperalgesia, thermal hyperalgesia or fantasy pain.

(2. Treatment of tactile abnormalities)
The term “tactile dysfunction” typically refers to a condition in a subject in which pain is induced by a normally harmless skin irritation (eg, palpation). The invention features a method of treating a tactile abnormality in a subject.

  In one embodiment, haptic abnormalities are treated by administering an effective amount of neublastin polypeptide alone to the subject.

  In a second embodiment, the tactile dysfunction comprises an effective amount of neublastin polypeptide from the group consisting of opioids, antiarrhythmic agents, local analgesics, local anesthetics, antispasmodics, antidepressants, corticosteroids and NSAIDS. Treated by administering to the subject in combination with an effective amount of an analgesia-inducing compound selected. In a preferred embodiment, the analgesia-inducing compound is an antispasmodic agent. In another preferred embodiment, the analgesia-inducing compound is gabapentin ((1-aminomethyl) cyclohexaneacetic acid) or pregabalin (S-(+)-4-amino-3- (2-methylpropyl) butyric acid). is there.

(3. Treatment for reducing loss of pain sensitivity)
In another embodiment, the invention features a method for reducing loss of pain sensitivity in a subject suffering from neuropathy. In a preferred embodiment, the neuropathy is diabetic neuropathy. In a preferred embodiment, the loss of pain sensitivity is a loss of thermal pain sensitivity. The present invention contemplates both prophylactic and therapeutic treatments.

  In prophylactic treatment, a neublastin polypeptide is administered to a subject at risk of developing loss of pain sensitivity; such a subject is expected to be a subject with early-stage neuropathy. Treatment with neublastin in such an environment serves to preventively treat patients at risk.

  In therapeutic treatment, a neublastin polypeptide is administered to a subject who has experienced loss of pain sensitivity as a result of neuropathic distress; such a subject is expected to be a subject with late-stage neuropathy The Treatment with neublastin under such circumstances serves to rescue appropriate pain sensitivity in the subject.

(4. Treatment of virus infection and virus-related neuropathy)
Prophylactic treatment of infectious and viral neuropathies is contemplated. Prophylactic treatment is indicated after confirmation of viral infection and before the onset of neuropathic pain. During treatment, NBN polypeptides may be associated with neuropathic pain (leprosy, neuropathic pain associated with Lyme disease, viruses (especially herpesviruses, more particularly herpes zoster virus that can lead to postherpetic neuralgia), human immunity, Administered in order to prevent the appearance of neuropathic pain associated with infection by a deficiency virus (HIV), and a virus selected from the group consisting of papillomavirus). In an alternative embodiment, the NBN polypeptide, if it appears, is administered to reduce the severity of neuropathic pain.

  Symptoms of acute viral infection often include the appearance of a rash. Other symptoms include, for example, the development of persistent pain in the affected area of the body, which is a common complication of herpes zoster infection (zoster). Postherpetic neuralgia can persist for over a month and may appear months after any of the rash-like symptoms have disappeared. Postherpetic neuralgia is very severe and persistent and can be very resistant to treatment.

(5. Treatment of diabetic neuropathy)
Prophylactic treatment of diabetes-related neuropathy is contemplated. Prophylactic treatment of diabetic neuropathy begins after confirmation of the initial diagnosis of diabetes or diabetes-related symptoms and before the onset of neuropathic pain. Prophylactic treatment of diabetic neuropathy also begins when the subject determines that there is a risk of developing diabetes or diabetes related symptoms. During treatment, NBN polypeptides are administered to prevent the appearance of neuropathic pain or to reduce the severity of neuropathic pain when it occurs.

  The results in FIGS. 6A and 6B relate to the treatment of diabetic neuropathy in human patients in need of such treatment. As described in Example 6, prevention of thermal hypoalgesia and prevention and reversal of thermal hyperalgesia are contemplated.

(6. Dosage)
The foregoing method comprises administering to the subject, preferably systemically, a formulation comprising a dosage of between 1 μg / kg to 30,000 μg / kg subject body weight per dose of neublastin polypeptide. Contemplated: Preferably, this dosage is between 10 μg / kg to 10,000 μg / kg subject body weight per dose; most preferably 25 μg / kg to 3000 μg / kg subject body weight per dose Between.

  Various dosing regimens for the treatment or prevention of tactile abnormalities are contemplated. In one embodiment, a method of administering a formulation comprising a neublastin polypeptide to a subject comprises administering NBN between 1 μg / kg to 30,000 μg / kg subject body weight per dose. In another embodiment, the dosage is between 10 μg / kg and 30,000 μg / kg of subject body weight per dose. In a further embodiment, the dosage is between 10 μg / kg and 10,000 μg / kg subject body weight per dose. In different embodiments, the dosage is between 25 μg / kg to 10,000 μg / kg subject body weight per dose. In yet another embodiment, the dosage is between 25 μg / kg and 3,000 μg / kg subject body weight per dose. In the most preferred embodiment, the dosage is between 50 μg / kg to 3,000 μg / kg subject body weight per dose.

  Similarly, various dosing schemes for treatment to modulate loss of pain sensitivity are contemplated. The method of administering to a subject a formulation comprising a neublastin polypeptide comprises administering NBN between 1 μg / kg to 30,000 μg / kg subject body weight per dose; preferably the dosage is Between 10 μg / kg to 10,000 μg / kg subject body weight per dose; most preferably, the dosage is between 25 μg / kg to 3,000 μg / kg subject body weight per dose.

(7. Delivery)
The neublastin polypeptide used in the foregoing method is any suitable delivery system, preferably a delivery selected from the group consisting of intravenous delivery, intramuscular delivery, pulmonary delivery, subcutaneous delivery, and intraperitoneal delivery. It can be administered via a system, most preferably intramuscular delivery, intravenous delivery or subcutaneous delivery. The neublastin polypeptide used in the foregoing methods can also be administered via intrathecal delivery.

(8. Prescription)
The present invention also provides a novel pharmaceutical composition comprising a therapeutically effective amount of neublastin polypeptide suspended, dissolved or dispersed in a pharmaceutically acceptable carrier.

  For use in therapy, the polypeptides of the invention can be administered in any convenient form. In preferred embodiments, the polypeptides of the invention are incorporated into a pharmaceutical composition with one or more adjuvants, excipients, carriers and / or diluents, and the pharmaceutical compositions are known in the art. Prepared by one of ordinary skill in the art using conventional methods. Further details about techniques for formulation and administration can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Maack Publishing Co., Easton, PA). Acceptable diluents, carriers and excipients typically do not adversely affect recipient homeostasis, particularly electrolyte balance. Acceptable carriers can include biocompatible salts, inert salts or bioabsorbable salts, buffering agents, oligosaccharides or polysaccharides, polymers, viscosity improvers, preservatives, and the like. One exemplary carrier includes normal physiological saline (0.15M NaCl (pH 7.0-7.4)). Another exemplary carrier includes 50 mM sodium phosphate, 100 mM sodium chloride.

(9. Regimen)
The frequency of dosing for the neublastin polypeptide of the invention is within the skill and clinical judgment of the physician. Typically, dosing regimens are established by clinical trials that can achieve optimal dosing parameters. However, the practitioner may vary such dosing regimens according to the subject's age, health, weight, sex and medical condition. The frequency of dosing can also differ between acute and chronic treatment of neuropathy. Furthermore, the dosing frequency can be varied depending on whether the treatment is prophylactic or therapeutic.

(Example 1: Expression of neublastin polypeptide)
(Expression in A. E. coli)
For expression and purification in bacteria, the plasmid encoding rat neublastin was transformed into E. coli as a His-tagged fusion protein with an enterokinase cleavage site immediately adjacent to the start site of the mature 113 amino acid NBN sequence. It was expressed in E. coli. E. E. coli cells were grown in a 500 L fermentor and provided a frozen cell paste. E. E. coli cells were lysed in Manton Gaulin Press and rat NBN was recovered from the insoluble washed pellet fraction.

  NBN was extracted from the pellet with guanidine hydrochloride, refolded and the His tag removed using enterokinase. For further purification, the product was then chromatographed on Ni NTA agarose (Qiagen) and chromatographed on Bakerbond WP CBX cation exchange resin.

  The resulting product was analyzed by SDS-PAGE, size exclusion chromatography (SEC), reverse phase HPLC, matrix-assisted laser desorption / ionization mass spectrometry (MALD / IMS), peptide mapping, evaluation of activity in KIRA ELISA, And subjected to extensive characterization including determination of endotoxin content. The purity of the NBN product measured by SDS-PAGE and SEC was higher than 95%. The NBN product migrated as a dimer under non-reducing conditions and was consistent with its expected structure. The endotoxin content of this material is conventionally less than 1 EU / mg. The specific activity of NBN in the KIRA ELISA is about 10 nM. The product was formulated at 1 mg / mL (pH 6.5) in PBS. This material can be supplied as a frozen liquid, which is stored at -70 ° C.

  A similar expression system was also constructed for human NBN. From these constructs, human NBN was obtained from E. coli. expressed in E. coli.

(B. Expression in mammalian cells)
(Construction of plasmid pJC070.14) In order to express neublastin cDNA in Chinese hamster ovary cells, a cDNA fragment encoding the prepro form of human neublastin was inserted into the mammalian expression vector pEAG347 and plasmid pJC070. 14 was produced. pEAG347 is a tandem SV40 early and adenovirus major late promoter (derived from plasmid pAD2β; Norton and Coffin, Mol. Cell. Biol. 5: 281 (1985)), a unique NotI cloning site, followed by SV40 late transcription termination and poly A signal. (Derived from plasmid pCMVβ; MacGregor and Caskey, Nucl. Acids. Res. 17: 2365 (1989)). In addition, pEAG347 contains a pUC19-derived plasmid backbone and pSV2dhfr-derived dhfr (for MTX selection and amplification in transfected CHO cells).

  Plasmid pJC070.14 was constructed in two steps. First, a fragment encoding the prepro form of human neublastin is obtained from the plasmid pUbilZ-NBN from the oligonucleotide

およびＰＦＵポリメラーゼを用いるポリメラーゼ連鎖反応を使用して単離した。このフラグメントを、ｐＰＣＲ−Ｓｃｒｉｐｔ Ａｍｐ ＳＫ（＋）のＳｒｆ−１部位にクローン化して、プラスミドｐＪＣ０６９を作製した。第二工程において、部分Ｎｏｔ−１消化を、プラスミドｐＪＣ０６９において実行して、６８５ｂｐフラグメント（ニューブラスチン遺伝子を含む）を作製し、これを、プラスミドｐＥＡＧ３４７のＮｏｔ−１部位にクローン化して、プラスミドｐＪＣ０７０．１４を作製した。プラスミドｐＪＣ０７０．１４におけるニューブラスチン遺伝子の転写を、アデノウイルス主要後期プロモーターによって制御する。

And was isolated using the polymerase chain reaction with PFU polymerase. This fragment was cloned into the Srf-1 site of pPCR-Script Amp SK (+) to create plasmid pJC069. In the second step, partial Not-1 digestion is performed in plasmid pJC069 to generate a 685 bp fragment (including the neublastin gene), which is cloned into the Not-1 site of plasmid pEAG347 and .14 was produced. Transcription of the neublastin gene in plasmid pJC070.14 is controlled by the adenovirus major late promoter.

(Preparation of CHO cell line expressing neublastin) 200 μg of pJC070.14 was linearized by digestion with the restriction endonuclease Mlu-1. The DNA was extracted with phenol: chloroform: isoamyl alcohol (25: 24: 1) and precipitated with ethanol. Linearized DNA was resuspended in 20 mM Hepes (pH 7.05), 137 mM NaCl, 5 mM KCl, 0.7 mM Na ₂ HPO ₄ , 6 mM dextrose (HEBS) and by electroporation (280 V and 960 μF). Introduced into ~ 4E7 CHO dukx B1 (dhfr-) cells (p23). Following electroporation, the cells were returned to culture in α + modified Eagle's medium (MEM) supplemented with 10% fetal bovine serum (FBS) for 2 days. Cells were then trypsinized and re-plated (100,000 cells / plate) for 5 days in 100 mm dishes in α-MEM (lack of ribo- and deoxyribonucleosides) supplemented with 10% dialyzed FBS. . Subsequently, the cells were split at a density of 100,000 cells / 100 mm plate and selected in 200 nM methotrexate. Resistant colonies were picked and scaled up to 6-well plates; conditioned media from each clone was screened using the specific assay for neublastin described below. Twelve clones expressing the highest levels of neublastin were scaled up to T162 flasks and subsequently re-assayed. These CHO cell lines produced neublastin in the range of 25-50 ng / ml / day.

  Triple complex assay for neublastin The presence of neublastin was determined using a modified version of the triple complex assay described by Sanicola et al. (Proc Natl Acad Sci USA 94: 6238 (1997)). Evaluated in the culture medium of the strain supernatant.

  Similar mammalian expression constructs were made and similar studies were performed on both human and rat NBN. An in vivo study of NBN activity in rats was performed. Rat studies were performed almost exclusively with rat NBN.

Example 2: Efficacy of full-length neuroblastin in a neuroligated animal model of neuropathic pain-prevention of neuropathic pain
The prophylactic effect of neublastin against tactile allodynia and thermal hyperalgesia was studied in the Chung L5 / L6 spinal nerve ligation (“SNL”) model (Kim and Chung (1992), Pain 50: 355-363). Spargue-Dawley male rats (250-300 g) were divided into four groups. The first group of rats (n = 7) received sham surgery and received vehicle by subcutaneous injection. A second group of rats (n = 7) received sham surgery and received rat neublastin (1 mg / kg) by subcutaneous injection. A third group of rats (n = 7) underwent spinal nerve ligation and received vehicle by subcutaneous injection. A fourth group of rats (n = 7) received spinal nerve ligation and received neublastin (1 mg / kg) by subcutaneous injection. The vehicle consisted of 5 mM phosphate and 150 mM sodium chloride (pH 6.5). Neublastin or vehicle is injected 30 minutes before spinal nerve ligation or sham surgery, then after ligation or surgery (after SNL), 2, 4, 7, 9, 11 Injections on the eyes and on the 14th day. Using the Von Frey behavioral test (Chaplan et al. (1994), J. Neurosci. Meth. 53: 55-63) and the Hargreave behavioral test (Hargreaves et al. (1998) Pain 32: 77-88) Each response was monitored. These pain responses were monitored before spinal nerve ligation or sham surgery to establish a baseline response, and then daily for two weeks after surgery.

  The results are shown in FIGS. 1 and 2 as the mean ± standard error of the mean. Subcutaneous administration of neublastin (indicated by the downward arrow in FIGS. 1 and 2) is almost complete of both types of neuropathic pain (tactile, FIG. 1 and fever, FIG. 2) in rats with spinal nerve ligation. Led to normalization. In sham-operated rats, subcutaneous administration of neublastin did not significantly change tactile sensation (FIG. 1) or heat sensitivity (FIG. 2). In spinal nerve ligated rats, the effect of neublastin on heat sensitivity was first revealed 3 days after the start of neublastin administration, whereas the effect on tactile allodynia was initially 4-5 days after onset, it was revealed slightly later. The effect of neublastin on heat sensitivity reached a plateau about 7 to 8 days after the start of neublastin administration, while the effect on tactile allodynia appears on the plateau about 10 to 11 days after the start of neublastin administration. Reached. The effect of neublastin did not diminish during the 2-3 day dosing interval. In fact, there was substantial normalization of both pain behavior between the administration of neublastin on days 4 and 7, as measured on days 5 and 6. After administration of neublastin on day 11, the remaining maximum normalization of both pain behaviors remains constant, indicating that the normalizing effect of neublastin on neuropathic pain is at least 3 days. Suggested to be maintained.

(Example 3: Treatment of neuropathy using a truncated neublastin polypeptide)
The prophylactic effect of a polypeptide containing the carboxy-terminal 102 amino acids of mature rat neublastin in treating tactile allodynia and thermal hyperalgesia (two peripheral neuropathic conditions) has been demonstrated by Chung L5 / L6 spinal nerve ligation (“SNL”). ]) In the model.

  Sprague-Dale male rats (approximately 250-300 g) are divided into four groups. The first group of rats (n = 6) undergo sham surgery and receive vehicle by subcutaneous injection. A second group of rats (n = 6) undergo sham surgery and are administered shortened neublastin (1 mg / kg) by subcutaneous injection. A third group of rats (n = 6) undergo spinal nerve ligation and receive vehicle by subcutaneous injection. The fourth group (n = 6) receives spinal nerve ligation and is administered shortened neublastin (1 mg / kg) by subcutaneous injection. The vehicle contains 5 mM phosphate and 150 mM sodium chloride (pH 6.5). Abbreviated neublastin or vehicle is administered on days 0, 2, 4, 7, 9, 11, and 14. On day 0, shortened neublastin is injected 30 minutes before spinal nerve ligation or sham surgery. Pain response was determined by Chaplan et al. (1994), J. MoI. Neurosci. Meth. 53: 55-63 and Hargreaves et al. (1998), Pain 32: 77-88. These tests monitor tactile and thermal responses, respectively. These pain responses are monitored before spinal nerve ligation or sham surgery to establish a baseline response. The pain response is then monitored daily for 2 weeks after surgery.

  Subcutaneous administration of truncated neublastin is expected to lead to normalization of both types of neuropathic pain in rats with spinal nerve ligation. In sham-operated rats, subcutaneous administration of truncated neublastin is not expected to significantly alter either tactile or thermal sensitivity. The effect of neublastin on heat sensitivity is expected to become apparent about 3 days after administration of neublastin, but the effect on tactile allodynia is expected to become slightly slower and initially evident.

Example 4: Effect of Neublastin in a Neuroligated Animal Model of Neuropathic Pain-Reversal of Neuropathic Pain
The reversal effect of neublastin on tactile allodynia and thermal hyperalgesia was studied in the Chung L5 / L6 spinal nerve ligation (“SNL”) model. Sprague-Dawley male rats (270-275 g) were divided into four groups. The first group of rats (n = 8) received sham surgery and received vehicle by subcutaneous injection. A second group of rats (n = 8) received sham surgery and was administered rat neublastin (1 mg / kg) by subcutaneous injection. A third group of rats (n = 8) received spinal nerve ligation and received vehicle by subcutaneous injection. A fourth group of rats (n = 8) received spinal nerve ligation and administered rat neublastin (1 mg / kg) by subcutaneous injection. The vehicle consisted of 5 mM phosphate and 150 mM sodium chloride (pH 6.5). Using the Von Frey behavioral test (Chaplan et al. (1994), J. Neurosci. Meth. 53: 55-63) and the Hargreave behavioral test (Hargreaves et al. (1988), Pain 32: 77-88), respectively, The thermal response was monitored. These pain responses were monitored before spinal nerve ligation or sham surgery to establish a baseline response and then monitored daily for 2 weeks after this surgery. Neublastin or vehicle was injected 60 minutes before the behavioral test on days 3, 5, 7, 10, 12 and 14 after ligation or surgery (after SNL).

  The results are shown in FIGS. 3 and 4 as the mean ± standard error of the mean. Both types of neuropathic pain behavior (tactile allodynia is shown in FIG. 3 and thermal hyperalgesia is shown in FIG. 4) were fully developed by day 3, as expected. Subcutaneous administration of neublastin (as indicated by the downward arrow in FIGS. 3 and 4) is associated with both types of neuropathic pain (tactile sensation in FIG. 3 and fever in FIG. 4) in spinal nerve ligated rats. A nearly complete reversal resulted, and the haptic and thermal responses were normalized. In sham-operated rats, subcutaneous administration of neublastin did not significantly alter tactile sensitivity (FIG. 3) or heat sensitivity (FIG. 4). In rats with spinal nerve ligation, the effect of neublastin on heat sensitivity and tactile allodynia was first revealed 2 to 3 days after the start of neublastin administration. The effect of neublastin on heat sensitivity and tactile allodynia reached a plateau about 7-8 days after the start of neublastin administration. The effect of neublastin did not diminish during the 2-3 day interval between doses. In fact, there was substantial normalization of both pain behaviors during the administration of neublastin on days 3, 5, 7, and 10.

Example 5 Neublastin Efficacy in Streptozotocin Animal Model of Neuropathic Pain-Prevention of Loss of Pain Sensitivity
The prophylactic effect of neublastin against loss of heat sensitivity (thermal insensitivity) was studied in a streptozotocin (“STZ”) model of diabetic neuropathy. Sprague-Dawley female rats (250-275 g) were divided into 4 groups, each consisting of 10 animals. One group of rats did not receive STZ; these rats received vehicle by subcutaneous injection. Three groups of rats received a single injection of STZ (50 mg / kg in sterile saline) and were subsequently confirmed to be hyperglycemic by a spectrophotometric assay of blood samples. Of the three groups of hyperglycemic rats, the first group received vehicle by subcutaneous injection, the second group received rat neublastin (0.1 mg / kg) by subcutaneous injection, and the third Groups of rats were administered rat neublastin (1 mg / kg) by subcutaneous injection. The vehicle consisted of 5 mM phosphate and 150 mM sodium chloride (pH 6.5). Neublastin or vehicle was administered 3 times a week (Monday, Wednesday, Friday schedule) for 8 weeks and started at the time of STZ induction of hyperglycemia. The latency time of the thermal response was evaluated after 8 weeks as described in Calcutt et al. (2000), Anesthesiology 93: 1271-1278. Briefly, a radiant heat source is applied to the plantar surface of the foot so that the surface temperature rises from 30 ° C. to 38.5 ° C. in 20 seconds and between the onset of heating and the withdrawal of the foot. The latency time was measured to evaluate the thermal response latency. An increase in the latency of paw withdrawal indicates a loss of heat sensitivity (thermal insensitivity).

  The results are shown in FIG. 5 as mean ± standard error of the mean. As expected, at 8 weeks after STZ, the latency of paw withdrawal was increased in vehicle treated STZ rats (STZ vehicle) compared to normal rats, and heat pain desensitization was induced by STZ injection. showed that. Neublastin administration prevented an increase in heat response latency in hyperglycemic (STZ) rats. Both doses of neublastin (1 mg / kg and 0.1 mg / kg) almost completely normalized heat sensitivity after 8 weeks of administration 3 times a week. These results demonstrate that neublastin prevents thermal pain desensitization, a loss of heat sensitivity that occurs in the STZ rat model of diabetic neuropathy.

(Example 6: Prevention of heat pain desensitization and prevention and reversal of heat hyperalgesia by neublastin in streptozotocin rats)
Rat NBN113 was prepared as described in Example 1 by E. coli. Prepared in E. coli. As described in Example 5, the preventive effect of neublastin on loss of heat sensitivity (heat pain dullness) and the prevention and reversal of increased heat sensitivity (heat hyperalgesia) by neublastin were as described in Example 5 for diabetic neuropathy. The streptozotocin (“STZ”) rat model was studied. The dosage study described in Example 5 was confirmed and expanded. The results are shown in FIGS. 6A and 6B.

  The results in FIGS. 6A and 6B are shown as mean ± standard error of the mean. As expected, at 4 weeks after STZ, the latency of paw withdrawal was reduced in vehicle-treated STZ rats compared to normal rats, indicating that thermal hyperalgesia was induced by STZ injection. All doses of subcutaneous neublastin (0.03 mg / kg and 0.1 mg / kg) prevented thermal hyperalgesia in STZ rats after administration for 4 weeks 3 times a week, as shown in FIG. 6A. All doses of subcutaneous neublastin (0.03 mg / kg and 0.1 mg / kg) prevented hypothermia in STZ rats after administration over 8 weeks 3 times a week, as shown in FIG. 6B. Furthermore, as shown in FIG. 6B, subcutaneous neublastin (0.1 mg / kg) administered during the second 4 weeks of the 8-week treatment study (veh; 0.1 mg / kg NBN) was: In addition to reversing the apparent thermal hyperalgesia 4 weeks after STZ treatment, it also prevented the development of hypothermia in STZ rats at 8 weeks. These results indicate that neublastin prevents and reverses thermal hyperalgesia in the STZ rat model of diabetic neuropathy and the loss of heat sensitivity that neublastin causes in the STZ rat model of diabetic neuropathy (thermal pain insensitive) Demonstrate that prevent

Example 7: Neuroblastin efficacy in a neuroligated animal model of neuropathic pain-reversal of neuropathic pain is dose dependent
Rat NBN113 was prepared as described in Example 1 by E. coli. Prepared in E. coli. Analysis of dose-dependent reversal by NBN113 of tactile allodynia and thermal hyperalgesia was performed in rats as described in Example 4, in 0.03 mg / kg, 0.1 mg / kg, 0.6 mg / kg and 2 mg. This was done using the Chung L5 / L6 spinal nerve ligation (“SNL”) animal model with a NBN dose of / kg. The experimental results shown in FIGS. 3 and 4 were confirmed and expanded. The results are shown in FIGS.

  The results in FIGS. 7 and 8 are shown as mean ± standard error of mean. BL indicates the baseline response. Both types of neuropathic pain behavior (tactile allodynia is shown in FIG. 7 and thermal hyperalgesia is shown in FIG. 8) were fully developed by day 2, as expected. Subcutaneous administration of 2 mg / kg neublastin (NBN) three times a week (as indicated by the arrows) induced both types of neuropathic pain (tactile sensation in FIG. 7 and heat in rats with spinal nerve ligation). In FIG. 8), resulting in normalization of tactile and thermal responses. As shown in FIG. 7, the reversal of neublastin in spinal nerve ligation-induced tactile allodynia was dose-responsive. 0.1 mg / kg s. c. Or 0.6 mg / kg s. c. Neublastin (NBN) administered at 1 time partially reversed tactile allodynia after 9 (and 11) days of administration 3 times a week, while 2 mg / kg s. c. Of NBN significantly reversed tactile allodynia after 7 (and 9 and 11) days of administration 3 times a week, and tactile allodynia after administration of 9 and 11 days 3 times a week It was almost completely reversed. Furthermore, the mean reversal of spinal nerve ligation-induced tactile allodynia 14 days after SNL increases the neublastin subcutaneous dose from 0.1 mg / kg to 0.6 mg / kg and to 2 mg / kg It was bigger. 0.03 mg / kg s. c. Of neublastin (NBN) did not significantly reverse spinal nerve ligation-induced tactile allodynia during 11 days of administration 3 times a week.

  As shown in FIG. 8, neublastin (NBN) reversal of spinal nerve ligation (SNL) -induced thermal hyperalgesia was also dose-dependent. Administration of 0.1 mg / kg NBN significantly reversed thermal hyperalgesia after 9 (and 11) days of administration 3 times a week, and 0.6 mg / kg NBN 7 times 3 times a week. Significantly reversed thermal hyperalgesia after (and 9 and 11) days of administration, and 2 mg / kg NBN reduced thermal hyperalgesia after 4 (and 7, 9, and 11) days of administration 3 times a week. Significantly reversed. Furthermore, the mean reversal of spinal nerve ligation-induced thermal hyperalgesia 14 days after SNL increases the subcutaneous dose of neublastin from 0.1 mg / kg to 0.6 mg / kg and to 2 mg / kg It was bigger. 0.03 mg / kg s. c. NBN did not significantly reverse spinal nerve ligation-induced thermal hyperalgesia during 11 days of administration 3 times a week.

(Equivalent)
Although specific embodiments have been disclosed in detail herein, this has been disclosed by way of example for purposes of illustration and is not intended to be limiting with respect to the scope of the appended claims. Absent. In particular, various substitutions, changes and modifications may be made to the present invention without departing from the spirit and scope of the invention as defined by the claims.

FIG. 1 is a line plot showing almost complete prevention of tactile allodynia by subcutaneous neublastin (NBN) in rats with L5 / L6 spinal nerve ligation (SNL). FIG. 2 is a line plot showing almost complete prevention of thermal hyperalgesia by subcutaneous neublastin (NBN) in rats with L5 / L6 spinal nerve ligation (SNL). FIG. 3 is a line plot showing almost complete reversal of fully established tactile allodynia by subcutaneous neublastin (NBN) in rats with L5 / L6 spinal nerve ligation (SNL). FIG. 4 is a line plot showing an almost complete reversal of fully established thermal hyperalgesia by subcutaneous neublastin (NBN) in rats with L5 / L6 spinal nerve ligation (SNL). FIG. 5 is a bar graph showing an almost complete normalization of heat-sensitive hypothesis by subcutaneous neublastin (NBN) in rats with STZ (streptozotocin) induced neuropathy. 6A and 6B show thermal hyperalgesia due to subcutaneous neublastin (NBN) in rats with STZ-induced neuropathy at 4 weeks (FIG. 6A) and 8 weeks (FIG. 6B) after STZ (streptozotocin) treatment. FIG. 6B is a graphical representation showing the prevention of heat pain (FIG. 6A), prevention of thermal pain desensitization (FIG. 6B), and reversal of thermal hyperalgesia (FIG. 6B). FIG. 7 is a line plot showing dose-dependent neublastin reversal of fully established tactile allodynia by subcutaneous neublastin (NBN) in rats with L5 / L6 spinal nerve ligation (SNL). FIG. 8 is a line plot showing dose-dependent neublastin reversal of fully established thermal hyperalgesia by subcutaneous neublastin (NBN) in rats with L5 / L6 spinal nerve ligation (SNL).

Sequence listing

Claims (18)

A pharmaceutical composition for treating or preventing tactile allodynia and thermal hyperalgesia in a subject, the composition comprises neublastin polypeptide, the New blanking Las Chin polypeptide via systemic delivery A pharmaceutical composition, wherein the composition is administered to the subject and is formulated for administration via subcutaneous delivery.   2. The pharmaceutical composition of claim 1, wherein the tactile allodynia and thermal hyperalgesia are associated with postherpetic neuralgia, diabetic neuropathy, or sciatica.   2. The pharmaceutical composition of claim 1, wherein the tactile allodynia and thermal hyperalgesia are associated with infection of the subject by a virus.   The pharmaceutical composition according to claim 3, wherein the virus is selected from the group consisting of herpes virus, human immunodeficiency virus (HIV), papilloma virus.   2. The pharmaceutical composition of claim 1, wherein the tactile allodynia and thermal hyperalgesia are neuropathic pain associated with administration of a therapeutic agent.   The pharmaceutical composition according to claim 5, wherein the therapeutic agent is an anticancer agent.   The pharmaceutical composition according to claim 6, wherein the anticancer agent is selected from the group consisting of taxol, taxotere, cisplatin, nocodazole, vincristine, vindesine, and vinblastine.   The pharmaceutical composition according to claim 5, wherein the therapeutic agent is an antiviral agent.   9. The pharmaceutical composition of claim 8, wherein the antiviral agent is selected from the group consisting of ddI, DDC, d4T, foscanet, dapsone, metronidazole, and isoniazid.   2. The pharmaceutical composition of claim 1, wherein the tactile allodynia and thermal hyperalgesia are due to injury associated with trauma.   11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the neublastin polypeptide is modified with a derivative moiety so as to have an extended residence time or increased concentration in body fluids. .   12. The pharmaceutical composition according to claim 11, wherein the derivative moiety is a polyethylene glycol moiety.   18. The pharmaceutical composition according to claim 17, wherein the derivative moiety is selected from the group consisting of aliphatic esters, amides, N-acyl derivatives, or O-acyl derivatives.   14. The pharmacology of any one of claims 1 to 13, wherein the neublastin polypeptide comprises an amino acid sequence that exhibits neurotrophic activity and is at least 85% identical to amino acids 28-140 of SEQ ID NO: 2. Composition. The New Bed Las Chin polypeptide exhibits neurotrophic activity and comprising at least 90% amino acids that are identical to the amino acid 28 to 140 of SEQ ID NO: 2, pharmaceutical according to any one of claims 1 to 13 Composition. The New Bed Las Chin polypeptide exhibits neurotrophic activity and comprising at least 95% amino acids that are identical to the amino acid 28 to 140 of SEQ ID NO: 2, pharmaceutical according to any one of claims 1 to 13 Composition. The New Bed Las Chin polypeptide comprises an amino acid 42-140 of SEQ ID NO: 2, The pharmaceutical composition according to any one of claims 1 to 13. The New Bed Las Chin polypeptide comprises an amino acid 37-140 of SEQ ID NO: 2, The pharmaceutical composition according to any one of claims 1 to 13.

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